| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Department of Dermatology and Allergology, Allergie-Centrum-Charité, Charité – Universitätsmedizin Berlin, Berlin, Germany;
Department of Dermatology, University Hospital Würzburg, Würzburg, Germany;
Department of Dermatology, University Hospital Hamburg-Eppendorf, Hamburg, Germany;
Department of Dermatology, University Hospital Lübeck, Lübeck, Germany; and
|| Department of Dermatology, University Hospital Mainz, Mainz, Germany
1Correspondence: Department of Dermatology and Allergy, Charité – Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany. E-mail: marcus.maurer{at}charite.de
SPECIFIC AIMS
Mast cells (MCs) are widely recognized to contribute to the pathology of inflammatory conditions including allergies and autoimmune diseases. In addition, MCs have recently been shown to promote inflammatory responses that are beneficial to the host. For example, peritoneal MCs control bacterial infections by initiating protective innate immune responses to bacteria, at least in part, by releasing tumor necrosis factor (TNF)-
and recruiting neutrophils (PMNs).
Cutaneous inflammatory responses are also associated with the activation of MCs and because of their large repertoire of proinflammatory and growth-promoting mediators, MCs have been repeatedly hypothesized to contribute to the healing of skin wounds. During early wound healing (WH), the MC mediators histamine, serotonin, and TNF- are thought to contribute to the induction of localized coagulation, extravasation, and leukocyte recruitment, important features of the initial inflammatory phase of WH. Subsequently, MCs reportedly promote the proliferation of fibroblasts, endothelial cells, and keratinocytes during the proliferative phase of WH. Finally, MCs may be involved in tissue remodeling, the key feature of late WH responses.
Despite this extensive body of suggestive evidence in support of MC functions during WH, a role of MCs in this context, at least to our knowledge, has yet to be proven, and the relevance and mechanisms of MC effects on WH remain to be characterized in detail. Therefore, we have used the well-established in vivo model of genetically MC-deficient KitW/KitW-v mice and their reconstitution with functional MCs, to probe whether direct and conclusive evidence for MC functions during skin WH can be obtained. The specific aims were 1) to find out if murine MCs are involved in cutaneous WH in vivo; 2) to determine if MCs are activated (degranulate) in response to skin wounding; 3) to investigate the initial inflammatory events during cutaneous wound repair, particularly to assess MC effects on extravasation and influx of neutrophils, and 4) to characterize mechanisms, by which MCs may influence these events.
PRINCIPAL FINDINGS
1. Early wound healing is impaired in the absence of MCs
To compare the differences in skin wound closure between KitW/KitW-v mice and normal Kit+/+ mice, we measured the wound area of full-thickness back skin wounds at defined time points after wounding with a 6 mm biopsy punch (Fig. 1
A). Wound areas in Kit+/+ mice were found to decrease in size immediately after wounding. In contrast, wounds in MC-deficient KitW/KitW-v mice did not decrease, but slightly increased, in size during the first hours after wounding and wound sizes only started to decrease after 12 h postwounding. A significant reduction of wound area sizes was first achieved after 2 days. Significant differences in wound sizes of KitW/KitW-v mice and Kit+/+ mice were detectable at all time points assessed until day 6 after wounding. Moreover, the proliferation rate of basal keratinocytes was significantly reduced in KitW/KitW-v mice as compared with their wild-type (WT) littermates 24 h postwounding.
2. MCs degranulate in response to skin wounding
To test whether MCs are activated by skin wounding in vivo, we assessed the extent of MC degranulation in skin wounds of C57BL/6 mice 1 h after wounding by quantitative histomorphometry. The majority of skin MCs (73%) directly adjacent to the wound exhibited signs of extensive degranulation. The extent of MC activation was found to correlate inversely with the distance of MCs from the wound edge.
3. Vascular permeability and neutrophil recruitment after wounding are decreased in the absence of mast cells
As skin MC degranulation can result in increased vascular permeability, we measured extravasation of Evans blue after wounding in KitW/KitW-v and Kit+/+ mice. Kit+/+ mice showed markedly increased levels of Evans blue extravasation at sites of wounding as compared with control skin (Fig. 1B
). Notably, increases in vascular permeability in Kit+/+ skin wounds were found to be significantly larger than those in KitW/KitW-v mice. To test whether the impaired up-regulation of vascular permeability at skin wound sites in KitW/KitW-v mice is associated with a reduction in subsequent neutrophil (PMN) recruitment (compared with Kit+/+ mice), we measured levels of the PMN enzyme myeloperoxidase (MPO) in skin wounds of both genotypes (Fig. 1C
). Increase in MPO activity in wounded skin was detectable in both types of mice as early as 6 h after wounding, when values in Kit+/+ mice were already higher than in KitW/KitW-v mice; 12 h after wounding, MPO levels in Kit+/+ mice exhibited a maximum increase as compared with control skin. In contrast, MPO-levels of KitW/KitW-v mice increased to a much lesser extent. Levels of PMN-recruitment in KitW/KitW-v mice never reached Kit+/+ levels, at any of the time points studied.
4. The closure of skin wounds, the up-regulation of vascular permeability, and PMN recruitment after wounding are MC dependent
In addition to their profound MC deficiency, KitW/KitW-v mice, as a consequence of their loss of function c-kit mutations, are also sterile, anemic, lack melanocytes and interstitial cells of Cajal, and the Kit ligand, stem cell factor (SCF), is not able to adequately stimulate its cognate receptor on selected keratinocyte populations. Thus, in theory, impaired WH in KitW/KitW-v mice could be due to any or a combination of these Kit-dependent defects, and independent of MCs.
To test this possibility, we reconstituted KitW/KitW-v mice locally and selectively with bone marrow-derived cultured MCs (BMCMCs) and assessed these mice for wound closure, extravasation, and PMN accumulation. Notably, the adoptive transfer of functional MCs to KitW/KitW-v mice led to a complete normalization of wound closure (Fig. 1A
). Moreover, extravasation and PMN accumulation (Fig. 1B, C
) were found to be fully restored in MC-reconstituted KitW/KitW-v mice.
5. The inhibition of histamine, but not the absence of TNF-, results in delayed skin wound closure
To assess mechanistically whether the MC products histamine and/or TNF-, both of which promote extravasation and PMN recruitment, are involved in MC-dependent skin WH responses, we analyzed skin wound closure in mice genetically deficient for TNF- (TNF-–/– mice) and in C57BL/6 mice treated with selective histamine receptor antagonists. Surprisingly, we found that TNF-–/– mice exhibit accelerated wound closure as compared with TNF-+/+ (Fig. 2
A). In contrast, C57BL/6 mice treated with the selective histamine-H1-receptor antagonist dimethindene showed significantly delayed skin wound closure as compared to vehicle treated mice, whereas treatment with the H2-receptor antagonist did not affect WH (Fig. 2B,C
).
CONCLUSIONS AND SIGNIFICANCE
Here, we show that the early closure of murine skin wounds is impaired in the absence of MCs (genetically MC-deficient KitW/KitW-v mice). Moreover, we demonstrate that normal WH is MC dependent, i.e., that the local and selective repair of skin MC deficiency by the adoptive transfer of functional MCs leads to the complete normalization of wound closure.
Because we had observed that WH impairment in MC-deficient mice was most prominent during the initial response to wounding, we speculated that MCs in early skin wounds undergo activation and degranulation. Quantitative histomorphometric analyses of skin wound MCs indeed revealed a striking "activation gradient": The closer MCs are localized to the edge of a skin wound, the more activation and degranulation they exhibit. This observation suggests that the MC-activating signals induced by wounding are rapidly generated within the wound edge, which is subsequently exposed to high extracellular concentrations of proinflammatory MC mediators.
Next, we asked whether MCs contribute to extravasation and/or the recruitment (Fig. 3
) of PMNs, two key features of early skin WH responses. Plasma extravasation after wounding was markedly reduced in the absence of MCs yet was restored when the skin MC deficiency of KitW/KitW-v mice had been repaired by the local transfer of MCs before wounding. These findings show that MCs are responsible for increasing vascular permeability in early skin wounds. Interestingly, the absence of MCs did not completely abrogate extravasation in our experiments, suggesting that MCs are largely, but not entirely, responsible for this event. Next, we studied PMN influx into wounded skin areas and found this element of initial WH also to be MC dependent, i.e., the accumulation of neutrophils in skin wounds of MC-deficient KitW/KitW-v mice was fully restored by the adoptive transfer of MCs.
Likely mediators of MC-dependent extravasation and PMN recruitment include histamine and TNF-. Surprisingly, no defects in wound closure occurred in TNF- deficient mice subjected to skin wounding as compared with their WT controls. On the contrary, wound closure was found to be slightly improved in the absence of TNF-, which clearly demonstrates that TNF- does not promote MC-dependent WH.
Histamine, a potent vasoactive mediator in several inflammatory settings, is also stored in large amounts by cutaneous MCs and earlier reports had suggested this MC mediator to support cutaneous WH. Our findings from experiments using histamine antagonists in mice subjected to skin wounding support this notion: WH is impaired by dimethindene, an H1-receptor antagonist, but not ranitidine, an H2-receptor antagonist. Interestingly, wound closure in dimethindene-treated mice was impaired to a much lesser extent as compared with MC-deficient mice, suggesting that histamine is one of several MC mediators that accounts for delayed wound closure in the absence of MCs. The fact that WH impairment in dimethinden-treated mice peaked at 48 h after wounding, i.e., when KitW/KitW-v mice and Kit+/+ or MC-reconstituted KitW/KitW-v mice exhibited the most prominent difference in wound area size, indicates that WH-promoting histamine is largely derived from skin MCs.
Taken together, our data demonstrate that cutaneous WH responses in mice are critically controlled by activated MCs, which after skin wounding promote increased extravasation, PMN influx, and normal wound closure. In addition to our ongoing attempts to identify the key MC activating signals in wounded murine skin, the evident next step now is to explore the relevance of our findings in the context of WH in human skin and to probe whether skin WH in patients can be promoted, e.g., by topical application of selected MC secretagogues and/or MC products. Likewise, it will be important to test whether long-term systemic therapy with antihistamines, which rank among the most widely prescribed and consumed drugs on the market, have any negative effects on human skin WH, comparable with the WH-inhibitory effects we show here for a clinically widely used H1 antagonist in murine skin.
|
|
|
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5837fje
This article has been cited by other articles:
|
|
 |
|
  G. Bansal, J. A. DiVietro, H. S. Kuehn, S. Rao, K. H. Nocka, A. M. Gilfillan, and K. M. Druey RGS13 Controls G Protein-Coupled Receptor-Evoked Responses of Human Mast Cells J. Immunol., December 1, 2008; 181(11): 7882 - 7890. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Kanda and S. Watanabe Histamine enhances the production of human -defensin-2 in human keratinocytes Am J Physiol Cell Physiol, December 1, 2007; 293(6): C1916 - C1923. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Textor, A. H. Licht, J. P. Tuckermann, R. Jessberger, E. Razin, P. Angel, M. Schorpp-Kistner, and B. Hartenstein JunB Is Required for IgE-Mediated Degranulation and Cytokine Release of Mast Cells J. Immunol., November 15, 2007; 179(10): 6873 - 6880. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
